Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaI.

Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P4(3), with unit-cell parameters a = b = 66.40, c = 47.33 A. The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal. The rhenium-containing structure has been refined to a resolution of 2.5 A and the perrhenate ion binding sites and liganding residues have been identified.

Quorum-sensing signal binding results in dimerization of TraR and its release from membranes into the cytoplasm.

Promoter binding by TraR and LuxR, the activators of two bacterial quorum-sensing systems, requires their cognate acyl-homoserine lactone (acyl-HSL) signals, but the role the signal plays in activating these transcription factors is not known. Soluble active TraR, when purified from cells grown with the acyl-HSL, contained bound signal and was solely in dimer form. However, genetic and cross-linking studies showed that TraR is almost exclusively in monomer form in cells grown without signal. Adding signal resulted in dimerization of the protein in a concentration-dependent manner. In the absence of signal, monomer TraR localized to the inner membrane while growth with the acyl-HSL resulted in the appearance of dimer TraR in the cytoplasmic compartment. Affinity chromatography indicated that the N-terminus of TraR from cells grown without signal is hidden. Analysis of heterodimers formed between TraR and its deletion mutants localized the dimerization domain to a region between residues 49 and 156. We conclude that binding signal drives dimerization of TraR and its release from membranes into the cytoplasm.

Hierarchical gene regulatory systems arising from fortuitous gene associations: controlling quorum sensing by the opine regulon in Agrobacterium.

Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierarchy involving induction by the opines agrocinopines A and B and a quorum-sensing system. Regulation by the opines is mediated by the repressor AccR, while quorum sensing is effected by the transcriptional activator TraR and its ligand, the acyl-homoserine lactone signal molecule Agrobacterium autoinducer (AAI). These last two elements combine to activate expression of the tra system at high population densities. Sequence analysis indicated that traR is the fourth gene of an operon, which we named arc, that is transcribed divergently from accR. Complementation analysis of mutations in the genes 5' to traR showed that the other members of the arc operon are not required for conjugation. Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR. Deletion analysis showed that AccR-regulated expression of traR initiates from a promoter located in the intergenic region between accR and orfA, the first gene of the arc operon. Reverse transcriptase-polymerase chain reaction (RT-PCR) and primer extension analyses indicated that the arc transcript initiates upstream of orfA and proceeds uninterrupted through traR. These results are consistent with a model in which quorum sensing is subordinate to the opine regulon because traR has become associated with an operon controlled by the opine-responsive transcriptional regulator.

Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer.

N-Acylhomoserine lactone (acyl-HSL)-mediated gene expression, also called autoinduction, is conserved among diverse gram-negative bacteria. In the paradigm Vibrio fischeri system, bioluminescence is autoinducible, and the lux operon requires the transcriptional activator LuxR and the acyl-HSL autoinducer for expression. The production of the acyl-HSL signal molecule is conferred by the luxI gene, and luxR encodes the transcriptional regulator. We show here that Erwinia stewartii, the etiological agent of Stewart's wilt of sweet corn, synthesizes an acyl-HSL. Mass spectral analysis identified the signal molecule as N-(-3-oxohexanoyl)-L-homoserine lactone, which is identical to the V. fischeri autoinducer. We have cloned and sequenced the gene that confers acyl-HSL biosynthesis, called esaI, and the linked gene, esaR, that encodes a gene regulator. The two genes are convergently transcribed and show an unusual overlap of 31 bp at their 3' ends. Sequence analysis indicates that EsaI and EsaR are homologs of LuxI and LuxR, respectively. EsaR can repress its own expression but seems not to regulate the expression of esaI. The untranslated 5' region of esaR contains an inverted repeat with similarity to the lux box-like elements located in the promoter regions of other gene systems regulated by autoinduction. However, unlike the other systems, in which the inverted repeats are located upstream of the -35 promoter elements, the esaR-associated repeat overlaps a putative -10 element. We mutagenized the esaI gene in E. stewartii by gene replacement. The mutant no longer produced detectable levels of the acyl-HSL signal, leading to a concomitant loss of extracellular polysaccharide capsule production and pathogenicity. Both phenotypes were restored by complementation with esal or by exogenous addition of the acyl-HSL.

Genetic analysis of the agrocinopine catabolic region of Agrobacterium tumefaciens Ti plasmid pTiC58, which encodes genes required for opine and agrocin 84 transport.

The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacterium tumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy. acc is approximately 6 kb in size. It consists of at least five genes, accA through accE, as defined by complementation analysis using subcloned fragments and transposon insertion mutations of acc carried on different plasmids within the same cell. All five regions are required for agrocin 84 sensitivity, and at least four are required for agrocinopine and agrocin 84 uptake. The complementation results are consistent with the hypothesis that each of the five regions is separately transcribed. Maxicell experiments showed that the first of these genes, accA, encodes a 60-kDa protein. Analysis of osmotic shock fractions showed this protein to be located in the periplasm. The DNA sequence of the accA region revealed an open reading frame encoding a predicted polypeptide of 59,147 Da. The amino acid sequence encoded by this open reading frame is similar to the periplasmic binding proteins OppA and DppA of Escherichia coli and Salmonella typhimurium and OppA of Bacillus subtilis.

Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction.

Conjugal transfer of Ti plasmids from Agrobacterium donors to bacterial recipients is controlled by two types of diffusible signal molecules. Induction is mediated by novel compounds, called opines, that are secreted by crown gall tumours. These neoplasias result from infection of susceptible plants by virulent agrobacteria. The second diffusible signal, called conjugation factor, is synthesized by the donor bacteria themselves. Production of this factor is induced by the opine. Here we show that conjugation is regulated directly by a transcriptional activator, TraR, which requires conjugation factor as a coinducer to activate tra gene expression. TraR is a homologue of LuxR, the lux gene activator from Vibrio fischeri which also requires an endogenously synthesized diffusible coinducer. The two regulatory systems are related; the two activator proteins show amino-acid sequence similarities and the lux system cofactor, autoinducer, will substitute for conjugation factor in the TraR-dependent activation of Ti plasmid tra genes.

Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor.

The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gall tumors. The opines also induce Ti plasmid-encoded genes required by the bacteria for opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR. Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor. accR can encode a 28-kDa protein. This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera.

Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.

We have totally synthesized a gene that codes for rat hepatic cytochrome b5. The 5' flanking region was designed for efficient expression of this gene in Escherichia coli by incorporating an optimum ribosome binding site and spacer region. Both a soluble form, analogous to the protease-treated microsomal protein, as well as the complete cytochrome with hydrophobic membrane anchor, was constructed and expressed. Transformants with the gene for the soluble protein overproduce authentic cytochrome b5 to a level of 8% of the total cell protein. The complete cytochrome is expressed to a lesser extent with most of the protein found in the cell membrane fraction. This represents complete synthesis and bacterial expression of a mammalian metalloprotein gene. Cytochrome b5 is normally a six-coordinate low spin heme protein with histidine-39 and histidine-63 as axial ligands. We have replaced histidine-63 with a methionine residue by cassette mutagenesis, utilizing specific restriction enzyme sites engineered into the synthetic gene. The resultant protein has histidine-39 as sole axial ligand and is five-coordinate high spin in the ferric resting state, as indicated by optical and electron spin resonance spectroscopy. The ability to generate mutant cytochrome b5 in high yield is a crucial step in understanding heme protein folding, protein-protein recognition and binding, and biological electron transfer processes.